Immunomodulatory Properties of Taranjebin (Camel’s Thorn) Manna and Its Isolated Carbohydrate Macromolecules

Abstract

Taranjebin manna is a substance produced by Poophilus nebulosus Leth. (Aphrophoridae) larva that feed from host plant Alhagi maurorum (Leguminosae). In Persian ethnomedicine, it is used as an antipyretic, antiviral, antimicrobial, demulcent, and adaptogen. But it is contraindicated in acute fever and some infections. This controversy might be due to its immunomodulatory properties. This study evaluated immunomodulatory properties of Taranjebin and its macromolecules. Taranjebin solution was prepared as described in traditional literature. After dialysis and precipitation, the macromolecules were isolated on DEAE Sephadex A-25. The cytotoxic/proliferative properties of Taranjebin and its isolated macromolecules on human Jurkat E6.1 cells were investigated (15.62-1000 μg/mL) using WST-1 reagent. Three of 4 isolated acidic polysaccharides inhibited the proliferation of Jurkat cells in a dose-dependent manner at concentrations higher than 31.25 μg/mL (IC₅₀ range of 44.81-147.97 μg/mL). The crude aqueous Taranjebin solution had proliferative effects. These results indicate the immunomodulatory properties of Taranjebin.

Herbal mannas are the sweet secretions usually created by insects’ activities on some plant species. Several types of herbal manna have been administered by herbalists for different therapeutic properties in Persian traditional medicine. The most repeatedly described manna in mediaeval Persian literatures are Bidkhesht or Willow manna, Gesengebin or Astragalus manna, Oak manna, Shirkhesht, Tamarisk manna, Taranjebin (Terengebin) or Al hagi manna, Atraphaxis manna, and Trehala manna or Shekartighal. ¹ Despite wide administration of these types of manna by Persian traditional practitioners or by Iranian folk medicine, little has been done to evaluate their safety or to provide evidence for their claimed therapeutic properties. Also, despite the presence of some rare preliminary reports on their monosaccharides, the chemical structures of their constituents (especially the polymeric molecules) are mostly unknown. One of the most economically important manna in Persian herbal market is Taranjebin, which is also known as Persian manna, Terengebin, Tar-angabin, Al hagi manna, or camel’s thorn manna. In traditional Iranian and Islamic medicine, this manna is also mentioned with other names such as Asal al nada (Dew honey) and Oshtorangebin (camel honey). In English, this manna is known as Alhagi manna, Caspian manna, Merniabin manna, and Hedysarum manna, while in French it is called Manne de perse, Manne d’hedysarum, and Manna d’alhagi. ^1,2

It is a resinous, sweet, yellow-brown substance with a tear-like shape (1-3 mm) that is produced by the nutrition–secretion activity of Poophilus nebulosus Leth (Aphrophoridae) larva. ³ Their larva feed from the host plant Alhagi maurorum Medik (Syn: Alhagi persarum Boiss. & Buhse, Alhagi camelorum DC, Alhagi pseudalhagi (M. Bieb.) Fisch., and Hedysarum alhagi L.) from the family Leguminosae. The larvae consume some of the plant metabolites and transform it to the manna as a secretive waste substance that appears on the leaves and branches of the host plant. Some literature introduce Larinu spp as a producing insect, ⁴ but this is controversial and was rejected by Askarzadeh et al. ^5,6

Taranjebin has been mainly used as a mild laxative and in the treatment of neonatal jaundice in Persian ethnomedicine. There are several clinical and in vivo studies on its therapeutic effects on neonatal jaundice. ^{7 –9} It has been used as a sweetener, mild laxative, antitussive, and demulcent in sore throat, mild cholagogue, and antispasmodic, diuretic, detersive for bladder, motive for humors, and as a body warmer. It was suggested in traditional Persian literature for health maintenance and to treat mild fever. In Iranian ethnomedicine, this manna has been used for treatment of rubella. On the other hand, it was contraindicated in acute fever, measles, smallpox, dysentery, hematuria, and piles. The dosage of the manna varied from 20 to 150 g, which depends on ailment and patient conditions. ^{10 –12}

Controversial indications and contraindications could be observed for fever, infectious, and viral diseases such as measles in old literatures and today’s herbalist prescriptions. It seems that antipyretic and antiviral properties of the manna are possible because of the immunomodulatory properties of Taranjebin and also this might explain the controversies surrounding the mannas. The aim of this study is to evaluate the immunomodulatory properties of this manna and its macromolecules.

Materials and Methods

Materials

Taranjebin manna was collected in September from plants growing in Bushehr province by a herbalist. The manna was authenticated by an expert pharmacognosist, and its herbarium voucher specimen was on file at the Department of Pharmacognosy of Shiraz University of Medical Sciences, Shiraz, Iran.

Human Jurkat E6.1 cells (NCBI code: C121, Jurkat-FHCRC cell line) were purchased from Pasteur Institute of Iran. The cell proliferation reagent WST-1 was purchased from Roche Applied Sciences (Germany). Other materials, reagents, and solvents were of the highest available chemical purity and were purchased from Merck or Sigma-Aldrich.

Separation of Soluble and Insoluble Fractions

Taranjebin manna was cleaned by hand picking of the plants parts. The Taranjebin solution was prepared by dispersing 130.51 g crude manna in 160 mL boiling water. To separate the soluble and insoluble fractions, the dispersion was filtered using a nylon mesh filter (42 μm pore size). The prepared cooled dispersion was centrifuged (10 minutes, 8000 × g, 25°C). The soluble portion was separated from the insoluble material. Both the gel-forming insoluble and soluble portions were dialyzed against distilled water using dialysis tubes (cut off 2 kDa) for 24 hours to discard small molecules. ¹³

Isolation and Characterization of Water-Soluble Carbohydrate Biopolymers

To isolate water-soluble biopolymers, ethanol (4 volumes) was added to the soluble part of Taranjebin manna. The mixture was kept at 4°C overnight and then centrifuged at 5°C and 22378 × g for 30 minutes. ^13,14 The sediment was freeze-dried and applied to an ion exchange column (2 × 25 cm) packed with DEAE Sephadex A-25, which was equilibrated to pH 6.8 using NaH₂PO₄–Na₂HPO₄ 50 mM buffer and eluted with gradient solutions of 0.02, 0.05, 0.1, 0.3, 0.5, 1, 1.5, 2 M NaCl. ¹⁵ Fractions (8 mL) were collected at a rate of 1 drop/min, and the same fractions were pooled and freeze-dried.

Structural Characterization of Biopolymers

Total sugar content of polysaccharides was determined spectrophotometrically by phenol–sulfuric acid assay with glucose as a standard. The protein content of the carbohydrate macromolecules was evaluated by Bradford assay, and the uronic content was determined spectrophotometrically against standard galacturonic acid. ¹⁶ The presence of starch backbone for the polysaccharides was tested by adding 2 drops of an aqueous iodine–potassium iodide solution to the samples. Starch was used as a positive control, which gives a dark bluish color with the reagent. ¹⁷

Cell Viability by WST-1 Assay

Human Jurkat E6.1 cells were cultured in complete media (CM10 containing RPMI 1640 supplemented with 10% fetal calf serum, 100 U/mL penicillin, and 100μg/mL streptomycin) at 37°C in 5% CO₂ at 90% humidity. The cells were then seeded in 96-well flat-bottom plates at a density of 2 × 10⁴ cells per well and 100μL medium containing different concentrations of Taranjebin manna crude solution or each of the isolated carbohydrate biopolymer in CM10 medium (15.62, 31.25, 62.5, 125, 250, 500, and 1000 μg/mL) was added. After 24 hours, WST-1 reagent (10 μL) was added to each well and the absorbance (440 nm) was determined after 4 hours. Each test was performed in triplicate; background values from wells containing CM10 without cells were subtracted and the average values for the triplicates were calculated. Inhibition of cell proliferation was determined by calculating the relative absorption rate of treated cells to untreated cells and IC₅₀ (concentration of isolated biopolymer at which half the cell growth is inhibited) value was determined. Etoposide (20 μg/mL) was used as a positive control. ¹⁸

Statistics

Analysis of variance was used to analyze the data followed by the LSD-t post hoc test for multiple comparisons (Computer Statistical Package, SPSS 15). Results were expressed as mean ± SD, and differences were considered statistically significant at P < .05.

Results and Discussion

Characterization of Polysaccharide and Carbohydrate Macromolecules

As shown in Figure 1, 91.74% of Taranjebin manna was water soluble and 8.26% consisted of water-insoluble and gel-forming molecules.

Figure 1.

Procedure for biopolymer isolation from Taranjebin manna and their yields.

The water-soluble portion of Taranjebin manna was subjected to DEAE Sephadex A-25 column chromatography, which resulted in 4 isolated macromolecules with the yields of 2 to 25 mg (Figure 1).

Since water solubility has an important role on bioavailability and biological properties of biopolymers and macromolecules, ¹⁹ only the water-soluble macromolecules of Taranjebin manna were subjected to immunomodulatory assay and further structure elucidation studies.

Among the water-soluble macromolecules, macromolecule D had the highest yield.

No protein content was detected in the isolated biopolymers. The highest uronic acid content was detected in A and D. The ratio of glucose to galacturonic acid was 2.43 for A, 9.57 for B, 2.39 for C, and 2.50 for D. Using iodine–potassium iodide reagent revealed that none of the isolated macromolecules has a starch backbone. All the isolated macromolecules had galacturono-glycan (acidic heteroglycan containing galacturonic acid) structure.

Immunomodulatory Activities of Taranjebin Macromolecules

In this study, Jurkat cells were used to study the immunomodulatory activities of the isolated macromolecules of Taranjebin manna. ²⁰ In vitro cytotoxicity/proliferation effects of each of the isolated macromolecules as well as crude water-soluble fraction of Taranjebin manna were determined. Macromolecules A and B inhibited the proliferation of Jurkat cells at concentrations higher than 15.62 μg/mL; macromolecules C and D inhibited the proliferation of Jurkat cells at concentrations higher than 31.25 μg/mL. For all of the macromolecules, this inhibition was in a dose-dependent manner (Figure 2).

Figure 2.

The effects of isolated macromolecules (A, B, C, and D) and crude water-soluble fraction of Taranjebin manna on proliferation of Jurkat cells at different concentrations.

According to the IC₅₀ value, the order of sensitivity of the Jurkat cell line to the isolated macromolecules was C > A > B > D (Table 1).

Table 1.

IC₅₀ Values of Isolated Macromolecules of Taranjebin Manna on Jurkat Cell Line^a.

	Macromolecules
	A	B	C	D
IC50 (μg/mL)	143.78 ± 0.09	147.97 ± 0.04	44.81 ± 0.06	>1000

^aData are expressed as the mean ± standard deviation.

In this study, etoposide was used as the positive control, and even at the lowest concentration, the cytotoxicity was higher than 50% (IC₅₀ < 3.91 μg/mL).

According to the IC₅₀ value, macromolecule D showed no significant cytotoxicity (Table 1). In contrast, the crude water-soluble fraction of Taranjebin manna increased the proliferation of Jurkat cells at all tested concentrations, but there was a convex relationship between Jurkat cell viability and the Taranjebin manna crude fraction. The highest proliferation rate of Jurkat cells was observed when they were treated with 125 μg/mL of crude water-soluble fraction of Taranjebin manna (Figure 2).

Since this study was designed to investigate the immunomodulatory effects of Taranjebin manna and its macromolecules, we did not determine the exact chemical constituents of the smaller molecules in the aqueous extract of the manna. However, it seems that stimulatory effects of different smaller molecules in the crude water-soluble fraction antagonize the inhibitory effects of macromolecules on Jurkat cells. Aqueous extract of Taranjebin manna was reported to be rich in sucrose, glucose, melezitose, and micronutrients such as copper (23.31 ± 1.55 mg/kg), zinc (18.61 ± 1.33 mg/kg), and iron (781.59 ± 25.11 mg/kg), ²¹ which may have had a role in the observed proliferative effect of the crude water-soluble fraction of the manna on Jurkat cells.

As we know, there is no other report on the immunomodulatory or cytotoxic properties of Taranjebin manna or its isolated carbohydrate macromolecules; thus, it was not possible to compare the results of this study with others. But there are several reports on the beneficial effects of Taranjebin manna on lowering serum bilirubin or in neonate jaundice in vivo. ^7,22,23 Some of these studies were conducted on human neonates as clinical trials but they did not report any serious side effects or toxicity in neonates for oral administration of crude Taranjebin manna. ^8,22,23 Kazerani et al investigated the possible toxic effects of 10 days of oral administration of Taranjebin manna (0.6-4.8 g/kg/day) in Syrian mice. They evaluated serum urea, creatinine, total bilirubin, alkaline phosphatase, and alanine aminotransferase and reported that this manna did not have cytotoxic effects on any of these hepatic and renal parameters. ²⁴ These reports are somehow inconsistent with the results of the present study since the crude water-soluble fraction of Taranjebin manna also did not show any cytotoxic effects on Jurkat cell line.

On the other hand, Etebari et al evaluated the genotoxicity of aqueous extract of Taranjebin manna on HepG2 cell line by the comet assay. The manna was reported to be genotoxic at concentrations of 5 μg/mL, 25 mg/mL, 100 mg/mL, and 50 mg/mL but it was safe at lower concentrations. They concluded that this genotoxicity might be related to glycoside and mucilaginous compounds in the manna. ²⁵ This finding is inconsistent with the results of present study, since only isolated macromolecules (A, B, and C) of Taranjebin manna showed some degrees of cytotoxicity on Jurkat cells.

In another study by Upur et al, the effects of a traditional polyherbal formulation (Abnormal Savda Munziq), which contained Taranjebin manna as one of its ingredients, was evaluated on HepG2 cells. They observed inhibition in HepG2 cell growth as well as cellular protein and DNA and RNA synthesis inhibition. ²⁶ In this study, we did not investigate the mechanism of the observed cytotoxicity of the isolated macromolecules, but the investigating of the genotoxicity as a possible mechanism can be considered for future work.

All the isolated macromolecules (A, B, C, and D) in this study were acidic heteroglucan containing galacturonic acid with different ratios. Isolation of acidic polysaccharides was reported from Alhagi-honey. They introduced Alhagi-honey as a sweet powder that is prepared from the secretions of the leaves of Alhagi sparsifolia. ^{27 –29} Jian et al isolated an acidic polysaccharide that was composed of mannose, glucose, galactose, and galacturonic acid with a molar ratio of 1.1:1.9:3.9:2.1. The main backbone was reported to be composed of (1 → 4)-β-d-GalpA-(1 → 4)β-d-GalpA-(1 → 4)-β-d-Galp-(1 → 4)-β-d-Galp-(1 → 6)α-d-Glcp-(1 → 4)α-d-Glcp(1 → , while the side chain is composed of → 6)-α-d-Glcp and 2-CH₃-α-d-Man. ²⁹ In another report by Jian et al, it was mentioned that 11 polysaccharides were isolated from Alhagi-honey with different structures and molecular weights. ³⁰ They did not discuss how Alhagi-honey is produced by the plant A sparsifolia. They also did not mention any possible role for insects in the production of the Alhagi-honey they investigated. Thus, we cannot be sure that if the polysaccharide they have purified can be found in Taranjebin manna.

Goncharov et al isolated a polysaccharide fraction from Alhagi maurorum with the monosaccharide composition of galactose, arabinose, and uronic acids. ³¹ Although these polysaccharides were reported from the plants in the Alhagi genus, the presence of acidic polysaccharides are somehow inconsistent with the results of present study.

We could not find any report on the immunomodulatory or cytotoxic properties of this manna but acidic glucan from other sources such as Ganoderma lucidum ³² or Grifola frondoscfi ³³ were reported to have antitumor and immunomodulatory properties. Due to diversity in cell lines used, investigated concentrations, molecular weight, and structures of the polysaccharides in these reports, it is difficult to compare the results. In addition, different mechanism were suggested for these polysaccharides including boosting host immune system ^34,35 or direct toxicity or genotoxicity on tumor cells. ^36,37

On the other hand, the cell line that was applied in this study (Jurkat cells) is an immortalized human T lymphocyte that phenotypically resembles resting human T lymphocytes. This cell line not only has been used in many reports as a target for testing the effects of new antitumor compounds but also is usually used to investigate the physiology and stimulation of T cells. ^21,38,39

Considering the nature of Jurkat cells, the high IC₅₀ (in comparison to etoposide) and the proliferative properties of the crude water-soluble fraction of Taranjebin manna, it can be suggested that in order to claim immunomodulatory or anticancer properties, the effects of crude aqueous fraction and each of its isolated macromolecule should be evaluated on normal human peripheral white blood cells as well as tumor cells originated from other lineages.

Conclusion

The results of present study showed that the total aqueous fraction of Taranjebin manna has immunostimulatory effects. In addition, the water-soluble fraction of Taranjebin manna contained several carbohydrate macromolecules with different biological activities and structure. Three of the isolated macromolecules showed some degree of cytotoxicity in a dose-dependent manner. In contrast, the crude water-soluble fraction of Taranjebin manna had proliferative effects, which indicate that smaller molecules in the manna antagonize the cytotoxic effects of the macromolecules. This is important especially because the crude aqueous fraction of Taranjebin manna is administered in neonates and infants as a remedy for jaundice and constipation.